One-step enzymatic and amine detection technique

ABSTRACT

A technique for detecting the presence or quantity of an enzyme (or enzyme inhibitor) and/or an amine within a test sample is provided. For example, in one embodiment, a diagnostic test kit is employed that utilizes reactive complexes that each includes a substrate joined (e.g., covalently bonded, physically adsorbed, etc.) to a reporter and a separation species. Upon contacting the reactive complexes, enzymes may cleave the substrate and release the reporter. Moreover, the test kit may also employ a chemichromic dye, i.e., a dye that exhibits a detectable color change upon chemical reaction with one or more functional groups, such as amino groups. The signal generated (directly or indirectly) by the reporter and chemichromic dye may then be used to indicate the presence or quantity of an enzyme (or enzyme inhibitor) and amine, respectively, within the test sample.

BACKGROUND OF THE INVENTION

The rapid diagnosis of infection is becoming increasingly important toimproving the effectiveness of subsequent treatment. Vaginal infection(“vaginitis”), for example, exists in three primary forms, i.e.,bacterial vaginosis, candidal vaginitis (“yeast”), and trichomonasvaginitis (“trich”). Various techniques have been developed in anattempt to rapidly diagnose individual forms of vaginitis. Bacterialvaginosis, for example, has been diagnosed using “clue cells” (vaginalepithelial cells with adherent surface bacteria). However, conventionaltechniques for confirming the presence of “clue cells” are oftencomplicated and slow. Techniques have been utilized that detect anelevated pH level in an infected sample.

Bacterial vaginosis and trichomonas vaginitis (primarily caused by theprotozoan, Trichomonas vaginalis) may also cause a “fishy” odor thatstems from an elevated level of amines, such as putrescine(1,4-diaminobutane), cadaverine (1,5-diamino pentane), trimethylamine,etc., in an infected vaginal sample. In bacterial vaginosis, forinstance, amines are believed to be produced by members of anaerobicbacteria, prevotella, bacteroides, mobiluncus, and peptococcus. Oneconventional test for detecting the presence of amines in a vaginal testsample is known as the “Whiff test”, which involves adding a strongalkali to a sample to form an enhanced odor. Unfortunately, such testsare undesired in that they require performance by a professional andutilize caustic chemicals. Another conventional technique for detectingamines in a sample is described in U.S. Pat. No. 5,124,254 to Hewlins,et al. Hewlins, et al. uses a diamine oxidase that reacts with diamines,such as putrescine and cadaverine, to give hydrogen peroxide. Thehydroxen peroxide is then detected by a chromogenic system.

Various attempts at diagnosing candidal vaginitis, which is primarilycaused by the presence of the yeast, Candida albicans, have also beendeveloped. One such method involves detecting the presence of enzymesthought to act as a virulence factor for Candida albicans, such asproteases (e.g., aspartic protease) and/or peptidases (e.g.,metallopeptidases). For example, U.S. Pat. No. 5,585,273 to Lawrence, etal. describes an enzyme assay for detecting Candida albicans asparticprotease. In Lawrence, et al., a sample, e.g., vaginal fluid, iscontacted with a solid support having a reporter enzyme immobilizedthereon. The reporter enzyme is releasable from the solid support uponaction of an enzymatically active aspartic protease. After contactingthe solid support, the sample is combined with an indicator susceptibleto a visible or detectable change upon action of the reporter enzyme. Ifthe indicator undergoes a detectable change, enzymatically activeaspartic protease is present.

Unfortunately, the techniques described above suffer from significantdisadvantages. One significant disadvantage is that many conventionaltechniques are unable to detect multiple forms of infection in a singletest sample. In addition, many conventional techniques are too slow,costly, and complex for ordinary consumer use. As such, a need currentlyexists for a technique for detecting multiple forms of infection in asingle test sample, which is fast, inexpensive, and easy to use.

SUMMARY OF THE INVENTION

In accordance with one embodiment of the present invention, a diagnostictest kit for detecting an amine, enzyme, or enzyme inhibitor within atest sample (e.g., vaginal fluid) is disclosed. The kit comprises aplurality of reactive complexes that each comprises a substrate joinedto a reporter and a separation species. The substrate is cleavable by anenzyme to release the reporter. The kit further comprises achromatographic medium that defines a first enzyme detection zone withinwhich an enzyme detection signal is capable of being generated. Thepresence or quantity of an enzyme, or an inhibitor thereof, isdeterminable from the enzyme detection signal. The chromatographicmedium further defines an amine detection zone within which is containeda chemichromic dye. The chemichromic dye is capable of undergoing acolor change in the presence of an amine, wherein the presence orquantity of an amine is determinable from the color change.

In accordance with another embodiment of the present invention, a methodfor detecting an amine, enzyme, or enzyme inhibitor within a test sampleis disclosed. The method comprises: i) contacting the test sample with achromatographic medium, the chromatographic medium defining an enzymedetection zone and an amine detection zone, wherein an enzyme detectionsignal is capable of being generated within the enzyme detection zoneand an amine detection signal is capable of being generated within theamine detection zone; ii) determining the presence or quantity of anenzyme or enzyme inhibitor from the enzyme detection signal; and iii)determining the presence or quantity of an amine from the aminedetection signal.

Other features and aspects of the present invention are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, directed to one of ordinary skill in the art, is setforth more particularly in the remainder of the specification, whichmakes reference to the appended figures in which:

FIG. 1 is a perspective view of one embodiment of an assay device thatmay be used in the diagnostic test kit of the present invention;

FIG. 2 is a graphical illustration of one embodiment for covalentlybonding a reporter to a substrate; and

FIG. 3 is a schematic illustration of one assaying technique that may beused in one embodiment of the present invention to detect the presenceor quantity of an amine and an enzyme (or enzyme inhibitor) within atest sample.

Repeat use of reference characters in the present specification anddrawings is intended to represent same or analogous features or elementsof the invention.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS Definitions

As used herein, the term “test sample” generally refers to a materialsuspected of containing an enzyme, enzyme inhibitor, and/or amine ofinterest. For example, the test sample may be obtained or derived from abiological source, such as a physiological fluid, including, blood,interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid,sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritonealfluid, vaginal fluid, amniotic fluid, and so forth. Besidesphysiological fluids, other liquid samples may be used such as water,food products, and so forth, for the performance of environmental orfood production assays. In addition, a solid material may be used as thetest sample. The test sample may be used directly as obtained from asource or following a pretreatment to modify the character of thesample. For example, such pretreatment may include preparing plasma fromblood, diluting viscous fluids, and so forth. Methods of pretreatmentmay also involve filtration, precipitation, dilution, distillation,mixing, concentration, inactivation of interfering components, theaddition of reagents, etc. Moreover, it may also be beneficial to modifya solid test sample to form a liquid medium, to release the enzyme,enzyme inhibitor, amine, etc.

DETAILED DESCRIPTION

Reference now will be made in detail to various embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, may be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncovers such modifications and variations as come within the scope of theappended claims and their equivalents.

In general, the present invention is directed to a technique fordetecting the presence or quantity of an enzyme (or enzyme inhibitor)and/or an amine within a test sample. For example, in one embodiment, adiagnostic test kit is employed that utilizes reactive complexes thateach includes a substrate joined (e.g., covalently bonded, physicallyadsorbed, etc.) to a reporter and a separation species. Upon contactingthe reactive complexes, enzymes may cleave the substrate and release thereporter. Moreover, the test kit may also employ a chemichromic dye,i.e., a dye that exhibits a detectable color change upon chemicalreaction with one or more functional groups, such as amino groups. Thesignal generated (directly or indirectly) by the reporter andchemichromic dye may then be used to indicate the presence or quantityof an enzyme (or enzyme inhibitor) and amine, respectively, within thetest sample.

I. Enzyme or Enzyme Inhibitor Detection

Various types of enzymes may be detected in accordance with the presentinvention. For instance, transferases, hydrolases, lyases, and so forth,may be detected. In some embodiments, the enzyme of interest is a“hydrolase” or “hydrolytic enzyme”, which refers to enzymes thatcatalyze hydrolytic reactions. Examples of such hydrolytic enzymesinclude, but are not limited to, proteases, peptidases, lipases,nucleases, homo- or hetero-oligosaccharidases, homo- orhetero-polysaccharidases, phosphatases, sulfatases, neuraminidases andesterases. In one embodiment, for example, peptidases may be detected.“Peptidases” are hydrolytic enzymes that cleave peptide bonds found inshorter peptides. Examples of peptidases include, but are not limitedto, metallopeptidases; dipeptidylpeptidase I, II, or IV; and so forth.In another embodiment, proteases may be detected. “Proteases” arehydrolytic enzymes that cleave peptide bonds found in longer peptidesand proteins. Examples of proteases that may be detected according tothe present invention-include, but are not limited to, serine proteases(e.g., chymotrypsin, trypsin, elastase, PSA, etc.), aspartic proteases(e.g., pepsin), thiol proteases (e.g., prohormone thiol proteases),metalloproteases, acid proteases, and alkaline proteases. Still otherenzymes are described in U.S. Pat. No. 6,243,980 to Bronstein, et al.and 2004/0081971 to Yue et al., which are incorporated herein in theirentirety by reference thereto for all purposes.

Likewise, any of a variety of known enzyme inhibitors may also bedetected in accordance with the present invention. For example, knowninhibitors of hydrolytic enzymes include, but are not limited to,inhibitors of proteases, peptidases, lipases, nucleases, homo- orhetero-oligosaccharidases, homo- or hetero-polysaccharidases,phosphatases, sulfatases, neuraminidases and esterases. Proteaseinhibitors may include, for instance, aspartic protease inhibitors,serine protease inhibitors, thiol protease inhibitors, metalloproteaseinhibitors, acid or alkaline protease inhibitors, and so forth. Somespecific examples of protease inhibitors include benzamideine, indole,pepstatin, ovomacroglobulin, haloperidol, transition state mimetics, andso forth.

Various techniques may generally be employed to detect the presence orquantity of an enzyme or enzyme inhibitor. For example, in someembodiments, reactive complexes are employed that each includes asubstrate joined to a reporter and a separation species. The term“substrate” generally refers to a substance that is chemically actedupon by an enzyme to form a product. The substrate may occur naturallyor be synthetic. Some suitable substrates for hydrolytic enzymesinclude, for instance, esters, amides, peptides, ethers, or otherchemical compounds having an enzymatically-hydrolyzable bond. Theenzyme-catalyzed hydrolysis reaction may, for example, result in ahydroxyl or amine compound as one product, and a free phosphate,acetate, etc., as a second product. Specific types of substrates mayinclude, for instance, proteins or glycoproteins, peptides, nucleicacids (e.g., DNA and RNA), carbohydrates, lipids, esters, derivativesthereof, and so forth. For instance, some suitable substrates forpeptidases and/or proteases may include peptides, proteins, and/orglycoproteins, such as casein (e.g., β-casein, azocasein, etc.), albumin(e.g., bovine serum albumin (BSA)), hemoglobin, myoglobin, keratin,gelatin, insulin, proteoglycan, fibronectin, laminin, collagen, elastin,and so forth. Still other suitable substrates are described in U.S. Pat.Nos. 4,748,116 to Simonsson, et al.; U.S. Pat. No. 5,786,137 to Diamond,et al.; U.S. Pat. No. 6,197,537 to Rao, et al.; and U.S. Pat. No.6,235,464 to Henderson, et al.; U.S. Pat. No. 6,485,926 to Nemori, etal., which are incorporated herein in their entirety by referencethereto for all purposes.

The reporters may contain any substance capable of directly orindirectly generating a detectable signal. Suitable detectablesubstances may include, for instance, chromogens; luminescent compounds(e.g., fluorescent, phosphorescent, etc.); radioactive compounds; visualcompounds (e.g., latex or metallic particles, such as gold); liposomesor other vesicles containing signal-producing substances; enzymes and/orsubstrates, and so forth. For instance, some enzymes suitable for use asdetectable substances are described in U.S. Pat. No. 4,275,149 toLitman, et al., which is incorporated herein in its entirety byreference thereto for all purposes. One example of an enzyme/substratesystem is the enzyme alkaline phosphatase and the substrate nitro bluetetrazolium-5-bromo-4-chloro-3-indolyl phosphate, or derivative oranalog thereof, or the substrate 4-methylumbelliferyl-phosphate. Othersuitable reporters may be described in U.S. Pat. Nos. 5,670,381 to Jou,et al. and U.S. Pat. No. 5,252,459 to Tarcha, et al., which areincorporated herein in their entirety by reference thereto for allpurposes.

In some embodiments, the reporters may contain a luminescent compoundthat produces an optically detectable signal. The luminescent compoundmay be a molecule, polymer, dendrimer, particle, and so forth. Forexample, suitable fluorescent molecules may include, but not limited to,fluorescein, europium chelates, phycobiliprotein, rhodamine, and theirderivatives and analogs. Other suitable fluorescent compounds aresemiconductor nanocrystals commonly referred to as “quantum dots.” Forexample, such nanocrystals may contain a core of the formula CdX,wherein X is Se, Te, S, and so forth. The nanocrystals may also bepassivated with an overlying shell of the formula YZ, wherein Y is Cd orZn, and Z is S or Se. Other examples of suitable semiconductornanocrystals may also be described in U.S. Pat. Nos. 6,261,779 toBarbera-Guillem, et al. and U.S. Pat. No. 6,585,939 to Dapprich, whichare incorporated herein in their entirety by reference thereto for allpurposes.

Further, suitable phosphorescent compounds may include metal complexesof one or more metals, such as ruthenium, osmium, rhenium, iridium,rhodium, platinum, indium, palladium, molybdenum, technetium, copper,iron, chromium, tungsten, zinc, and so forth. Especially preferred areruthenium, rhenium, osmium, platinum, and palladium. The metal complexmay contain one or more ligands that facilitate the solubility of thecomplex in an aqueous or nonaqueous environment. For example, somesuitable examples of ligands include, but are not limited to, pyridine;pyrazine; isonicotinamide; imidazole; bipyridine; terpyridine;phenanthroline; dipyridophenazine; porphyrin, porphine, and derivativesthereof. Such ligands may be, for instance, substituted with alkyl,substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino,hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide,sulfur-containing groups, phosphorus containing groups, and thecarboxylate ester of N-hydroxy-succinimide.

Porphyrins and porphine metal complexes possess pyrrole groups coupledtogether with methylene bridges to form cyclic structures with metalchelating inner cavities. Many of these molecules exhibit strongphosphorescence properties at room temperature in suitable solvents(e.g., water) and an oxygen-free environment. Some suitable porphyrincomplexes that are capable of exhibiting phosphorescent propertiesinclude, but are not limited to, platinum (II) coproporphyrin-I and III,palladium (II) coproporphyrin, ruthenium coproporphyrin,zinc(II)-coproporphyrin-I, derivatives thereof, and so forth. Similarly,some suitable porphine complexes that are capable of exhibitingphosphorescent properties include, but not limited to, platinum(II)tetra-meso-fluorophenylporphine and palladium(II)tetra-meso-fluorophenylporphine. Still other suitable porphyrin and/orporphine complexes are described in U.S. Pat. Nos. 4,614,723 to Schmidt,et al.; U.S. Pat. No. 5,464,741 to Hendrix; U.S. Pat. No. 5,518,883 toSoini; U.S. Pat. No. 5,922,537 to Ewart, et al.; U.S. Pat. No. 6,004,530to Sagner, et al.; and U.S. Pat. No. 6,582,930 to Ponomarev, et al.,which are incorporated herein in their entirety by reference thereto forall purposes.

Bipyridine metal complexes may also be utilized as phosphorescentcompounds. Some examples of suitable bipyridine complexes include, butare note limited to,bis[(4,4′-carbomethoxy)-2,2′-bipyridine]2-[3-(4-methyl-2,2′-bipyridine-4-yl)propyl]-1,3-dioxolaneruthenium (II);bis(2,2′bipyridine)[4-(butan-1-al)-4′-methyl-2,2′-bi-pyridine]ruthenium(II); bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine-4′-yl)-butyricacid]ruthenium (II); tris(2,2′bipyridine)ruthenium (II);(2,2′-bipyridine)[bis-bis(1,2-diphenylphosphino)ethylene]2-[3-(4-methyl-2,2′-bipyridine-4′-yl)propyl]-1,3-dioxolaneosmium (II);bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine)-butylamine]ruthenium(II);bis(2,2′-bipyridine)[1-bromo-4(4′-methyl-2,2′-bipyridine-4-yl)butane]ruthenium(II); bis(2,2′-bipyridine)maleimidohexanoic acid,4-methyl-2,2′-bipyridine-4′-butylamide ruthenium (II), and so forth.Still other suitable metal complexes that may exhibit phosphorescentproperties may be described in U.S. Pat. Nos. 6,613,583 to Richter, etal.; U.S. Pat. No. 6,468,741 to Massey, et al.; U.S. Pat. No. 6,444,423to Meade, et al.; U.S. Pat. No. 6,362,011 to Massey, et al.; U.S. Pat.No. 5,731,147 to Bard, et al.; and U.S. Pat. No. 5,591,581 to Massey, etal., which are incorporated herein in their entirety by referencethereto for all purposes.

In some cases, “time-resolved” luminescent detection techniques areutilized. Time-resolved detection involves exciting a luminescentcompound with one or more short pulses of light, then typically waitinga certain time (e.g., between approximately 1 to 100 microseconds) afterexcitation before measuring the remaining the luminescent signal. Inthis manner, any short-lived phosphorescent or fluorescent backgroundsignals and scattered excitation radiation are eliminated. This abilityto eliminate much of the background signals may result in sensitivitiesthat are 2 to 4 orders greater than conventional fluorescence orphosphorescence. Thus, time-resolved detection is designed to reducebackground signals from the emission source or from scattering processes(resulting from scattering of the excitation radiation) by takingadvantage of the characteristics of certain luminescent materials.

To function effectively, time-resolved techniques generally require arelatively long emission lifetime for the luminescent compound. This isdesired so that the compound emits its signal well after any short-livedbackground signals dissipate. Furthermore, a long luminescence lifetimemakes it possible to use low-cost circuitry for time-gated measurements.For example, the detectable compounds may have a luminescence lifetimeof greater than about 1 microsecond, in some embodiments greater thanabout 10 microseconds, in some embodiments greater than about 50microseconds, and in some embodiments, from about 100 microseconds toabout 1000 microseconds. In addition, the compound may also have arelatively large “Stokes shift.” The term “Stokes shift” is generallydefined as the displacement of spectral lines or bands of luminescentradiation to a longer emission wavelength than the excitation lines orbands. A relatively large Stokes shift allows the excitation wavelengthof a luminescent compound to remain far apart from its emissionwavelengths and is desirable because a large difference betweenexcitation and emission wavelengths makes it easier to eliminate thereflected excitation radiation from the emitted signal. Further, a largeStokes shift also minimizes interference from luminescent molecules inthe sample and/or light scattering due to proteins or colloids, whichare present with some body fluids (e.g., blood). In addition, a largeStokes shift also minimizes the requirement for expensive,high-precision filters to eliminate background interference. Forexample, in some embodiments, the luminescent compounds have a Stokesshift of greater than about 50 nanometers, in some embodiments greaterthan about 100 nanometers, and in some embodiments, from about 100 toabout 350 nanometers.

For example, one suitable type of fluorescent compound for use intime-resolved detection techniques includes lanthanide chelates ofsamarium (Sm (III)), dysprosium (Dy (III)), europium (Eu (III)), andterbium (Tb (IIII)). Such chelates may exhibit strongly red-shifted,narrow-band, long-lived emission after excitation of the chelate atsubstantially shorter wavelengths. Typically, the chelate possesses astrong ultraviolet excitation band due to a chromophore located close tothe lanthanide in the molecule. Subsequent to excitation by thechromophore, the excitation energy may be transferred from the excitedchromophore to the lanthanide. This is followed by a fluorescenceemission characteristic of the lanthanide. Europium chelates, forinstance, have exceptionally large Stokes shifts of about 250 to about350 nanometers, as compared to only about 28 nanometers for fluorescein.Also, the fluorescence of europium chelates is long-lived, withlifetimes of about 100 to about 1000 microseconds, as compared to about1 to about 100 nanoseconds for other fluorescent compound. In addition,these chelates have a narrow emission spectra, typically havingbandwidths less than about 10 nanometers at about 50% emission. Onesuitable europium chelate is N-(p-isothiocyanatobenzyl)-diethylenetriamine tetraacetic acid-Eu⁺³.

In addition, lanthanide chelates that are inert, stable, andintrinsically fluorescent in aqueous solutions or suspensions may alsobe used in the present invention to negate the need for micelle-formingreagents, which are often used to protect chelates having limitedsolubility and quenching problems in aqueous solutions or suspensions.One example of such a chelate is4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6-bis([N,N-bis(carboxymethyl)amino]methyl)-pyridine[Ref: Lovgren, T., et al.; Clin. Chem. 42, 1196-1201 (1996)]. Severallanthanide chelates also show exceptionally high signal-to-noise ratios.For example, one such chelate is a tetradentate β-diketonate-europiumchelate [Ref: Yuan, J. and Matsumoto, K.; Anal. Chem. 70, 596-601(1998)]. In addition to the fluorescent compounds described above, othercompounds that are suitable for use in the present invention may bedescribed in U.S. Pat. Nos. 6,030,840 to Mullinax, et al.; U.S. Pat. No.5,585,279 to Davidson; U.S. Pat. No. 5,573,909 to Singer, et al.; U.S.Pat. No. 6,242,268 to Wieder, et al.; and U.S. Pat. No. 5,637,509 toHemmila, et al., which are incorporated herein in their entirety byreference thereto for all purposes.

As stated, the reporter may indirectly generate a detectable signal insome embodiments of the present invention. In such instances, thereporter may not specifically contain a detectable substance, butinstead be capable of interacting with a detectable substance togenerate a detection signal. For example, in some embodiments, thereporter may be a member of a specific binding pair, i.e., two differentmolecules where one of the molecules chemically and/or physically bindsto the second molecule. Immunoreactive specific binding members mayinclude antigens, haptens, antibodies (primary or secondary), andcomplexes thereof, including those formed by recombinant DNA methods orpeptide synthesis. An antibody may be a monoclonal or polyclonalantibody, a recombinant protein or a mixture(s) or fragment(s) thereof,as well as a mixture of an antibody and other specific binding members.The details of the preparation of such antibodies and their suitabilityfor use as specific binding members are well known to those skilled inthe art. Other common specific binding members include, but are notlimited to, biotin and avidin, streptavidin, neutravidin, captavidin, oran anti-biotin antibody; protein A and G; carbohydrates and lectins,complementary nucleotide sequences (including probe and capture nucleicacid sequences used in DNA hybridization assays to detect a targetnucleic acid sequence); complementary peptide sequences including thoseformed by recombinant methods; effector and receptor molecules; hormoneand hormone binding protein; enzyme cofactors and enzymes, enzymeinhibitors and enzymes; derivatives thereof, and so forth. Furthermore,specific binding pairs may include members that are analogs,derivatives, and/or fragments of the original specific binding member.When used to indirectly generate a signal, a reporter that is a memberof a specific binding pair may be placed into contact with a probeconjugated with another member of the specific binding pair. Thus, thereleased reporter will bind to the conjugated probe, which may then bereadily detected (directly or indirectly) using techniques well known tothose skilled in the art.

Whether or not the reporter directly or indirectly generates a signal,it may contain particles (sometimes referred to as “beads” or“microbeads”). Among other things, particles enhance the ability of thereporter to travel through a chromatographic medium and becomeimmobilized within a detection zone, such as described below. Forinstance, naturally occurring particles, such as nuclei, mycoplasma,plasmids, plastids, mammalian cells (e.g., erythrocyte ghosts),unicellular microorganisms (e.g., bacteria), polysaccharides (e.g.,agarose), etc., may be used. Further, synthetic particles may also beutilized. For example, in one embodiment, latex particles are labeledwith a fluorescent or colored dye. Although any latex particle may beused, the latex particles are typically formed from polystyrene,butadiene styrenes, styreneacrylic-vinyl terpolymer,polymethylmethacrylate, polyethylmethacrylate, styrene-maleic anhydridecopolymer, polyvinyl acetate, polyvinylpyridine, polydivinylbenzene,polybutyleneterephthalate, acrylonitrile, vinylchloride-acrylates, andso forth, or an aldehyde, carboxyl, amino, hydroxyl, or hydrazidederivative thereof. Other suitable particles may be described in U.S.Pat. Nos. 5,670,381 to Jou, et al. and U.S. Pat. No. 5,252,459 toTarcha, et al. Commercially available examples of suitable fluorescentparticles include fluorescent carboxylated microspheres sold byMolecular Probes, Inc. under the trade names “FluoSphere” (Red 580/605)and “TransfluoSphere” (543/620), as well as “Texas Red” and 5- and6-carboxytetramethylrhodamine, which are also sold by Molecular Probes,Inc. of Eugene, Oreg. In addition, commercially available examples ofsuitable colored, latex microparticles include carboxylated latex beadssold by Bangs Laboratories, Inc. of Fishers, Ind.

When utilized, the shape of the particles may generally vary. In oneparticular embodiment, for instance, the particles are spherical inshape. However, it should be understood that other shapes are alsocontemplated by the present invention, such as plates, rods, discs,bars, tubes, irregular shapes, etc. In addition, the size of theparticles may also vary. For instance, the average size (e.g., diameter)of the particles may range from about 0.1 nanometers to about 1,000microns, in some embodiments, from about 0.1 nanometers to about 100microns, and in some embodiments, from about 1 nanometer to about 10microns. For instance, “micron-scale” particles are often desired. Whenutilized, such “micron-scale” particles may have an average size of fromabout 1 micron to about 1,000 microns, in some embodiments from about 1micron to about 100 microns, and in some embodiments, from about 1micron to about 10 microns. Likewise, “nano-scale” particles may also beutilized. Such “nano-scale” particles may have an average size of fromabout 0.1 to about 10 nanometers, in some embodiments from about 0.1 toabout 5 nanometers, and in some embodiments, from about 1 to about 5nanometers.

In addition to being joined to a reporter, such as described above, thesubstrate is also joined to a separation species. The separation speciesmay generally be any material that facilitates separation of releasedreporters from unreacted complexes. In some embodiments, for example,such separation is achieved through chemical binding. Specifically, theseparation species may be a specific binding member, such as describedabove, which may bind to another member of the binding pair toaccomplish the desired separation. When the reporter contains a specificbinding member, it is generally desired that it is different than andhas no specific binding affinity for the specific binding member of theseparation species.

Besides chemical binding, other separation techniques may also beutilized. For instance, in one embodiment, magnetic separationtechniques are employed. In such embodiments, the separation species maycontain a magnetic substance. Generally, a material is considered“magnetic” or “magnetically responsive” if it is influenced by theapplication of a magnetic field, such as, for example, if it isattracted or repulsed or has a detectable magnetic susceptibility orinduction. For instance, some examples of suitable magneticallyresponsive substances that may be used to impart magnetic propertiesinclude, but are not limited to, paramagnetic materials,superparamagnetic materials, ferromagnetic materials, ferrimagneticmaterials, and metamagnetic materials. Specific examples are metals suchas iron, nickel, cobalt, chromium, and manganese; lanthanide elements,such as neodymium, erbium; alloys, such as magnetic alloys of aluminum,nickel, cobalt, or copper; oxides, such as ferric oxide (Fe₃O₄), ferrousoxide (Fe₂O₃), chromium oxide (CrO₂), cobalt oxide (CoO), nickel oxide(NiO₂), or manganese oxide (Mn₂O₃); composite materials, such asferrites; and solid solutions, such as magnetite with ferric oxide. Insome embodiments of the present invention, the magnetic substancecontains a magnetic particle. When utilized, the shape and/or size ofthe particles may vary, such as described above.

The separation species and reporter may generally be attached to thesubstrate using any of a variety of well-known techniques. For instance,covalent attachment of a separation species and/or reporter to asubstrate may be accomplished using carboxylic, amino, aldehyde,bromoacetyl, iodoacetyl, thiol, epoxy and other reactive functionalgroups, as well as residual free radicals and radical cations, throughwhich a coupling reaction may be accomplished. A surface functionalgroup may also be incorporated as a functionalized co-monomer becausethe surface of the reporter may contain a relatively high surfaceconcentration of polar groups. In certain cases, the separation speciesand/or reporter may be capable of direct covalent bonding to a substratewithout the need for further modification. It should also be understoodthat, besides covalent bonding, other attachment techniques, such asphysical adsorption, may also be utilized in the present invention.Still other non-covalent linkage techniques may employ antibodies and/orantigens, such as secondary antibodies (e.g., avidin, streptavidin,neutravidin, and/or biotin).

One particular technique for covalently bonding a reporter andseparation species to a substrate will now be described in more detail.In this particular embodiment, the substrate is β-casein, the reporteris a dyed particle, and the separation species is a biotin derivative.For example, the reporter may be red carboxylated latex particlesavailable from Molecular Probes, Inc. under the name “FluoSphere.”Likewise, the separation species may besulfosuccinimidyl-6-(biotin-amido) hexanoate, which is available fromPierce Biotechnology, Inc. of Rockford, Ill. under the name EZ-Link®Sulfo-NHS-LC-Biotin. Techniques employed in making such NHS-activatedbiotins are believed to be described in U.S. Pat. No. 5,872,261 toBremmer, et al., which is incorporated herein in its entirety byreference thereto for all purposes.

To covalently conjugate the dyed particle with β-casein, the carboxylicgroups on the particle surface are first activated with a carbodiimide(e.g., ethylcarbodiimide hydrochloride (EDC)), such as shown in FIG. 2.Because protein and glycoprotein substrates (e.g., β-casein) typicallypossess primary amine groups (NH₂), such as on the side chain of lysine(K) residues and/or the N-terminus of each polypeptide, the activatedcarboxylic acid groups may then be reacted with the primary amine (—NH₂)groups of the substrate to form an amide bond. This reaction may occurin a buffer, such as phosphate-buffered saline (PBS) (e.g., pH of 7.2),2-(N-morpholino) ethane sulfonic acid (MES) (e.g., pH of 5.3), or boratebuffer (e.g., pH of 8.5). If desired, the resulting reactive complexesmay then be blocked with ethanolamine, for instance, to block anyremaining activated sites.

In a somewhat similar manner, the biotin-based separation species mayalso be covalently bonded to β-casein. For example, NHS-activatedbiotins may form covalent amide bonds with the primary amine groupspresent on the substrate (optionally in the presence of a buffer). Anexample of such a reaction is set forth below:

Once formed, a user may allow the test sample to incubate with thereactive complexes for a certain period of time. For example, thoseskilled in the art readily recognize that the time of incubation for anenzyme-catalyzed reaction depends on the activity of the enzyme ofinterest, which in turn depends on in part on the temperature, pH,substrate concentration, the presence of inhibitors (competitive (bindsto enzyme), uncompetitive (binds to enzyme-substrate complex), ornoncompetitive (binds to enzyme and/or enzyme-substrate complex)), andso forth. These factors may be selectively controlled as desired toincrease or decrease the incubation time. For example, the time forincubation may be greater than about 1 minute, in some embodiments fromabout 5 to about 50 minutes, and in some embodiments, from about 10 toabout 25 minutes. Likewise, the pH may be selectively controlled tofacilitate enzyme activity. For example, high levels of basic substances(e.g., amines) within a test sample may result in a pH that is too highfor optimum activity of some enzymes, e.g., greater than 8.Specifically, an enzyme may possess optimum activity at a pH level offrom about 3 to about 8, and in some embodiments, from about 4 to about7. Thus, if desired, a buffer or other pH-altering compound may beemployed to maintain the desired pH.

After incubation, any enzyme present within the test sample willtypically cleave the substrate of at least a portion of the reactivecomplexes. As a result, various materials may be formed, includingreleased reporters, released separation species, partially cleavedcomplexes (e.g., enzyme-reporter-substrate-separation species), andunreacted complexes (e.g., reporter-substrate-separation species).Longer incubation times and greater enzyme concentrations may result ina greater concentration of released reporters and separation species inthe resulting incubation mixture. Further, it should be understood thatthe “released” reporters and separation species may or may not containfragments of the complex depending on the nature of the substrate andenzyme. For instance, when using longer chain substrates (e.g.,proteins), the released reporters and separation species may containpeptide fragments from the protein substrate. On the other hand, whenusing shorter chain substrates (e.g., peptides), the released reportersand separation species may be relatively free of such fragments.

As stated above, various separation techniques may be utilized in thepresent invention for separating any released reporters from unreactedcomplexes including, but not limited to, chemical separation techniques,magnetic separation techniques, etc. In one particular embodiment, forexample, the diagnostic test kit contains an assay device that employs achromatographic medium for separating unreacted complexes from releasedreporters. In contrast to other techniques, such as centrifugation, theuse of a chromatographic medium may simplify and reduce the costs of theresulting diagnostic test kit for many consumer applications, includingthose in which a disposable kit is desired. Further, the use of achromatographic medium also provides for a mechanism in which twodifferent species, i.e., an enzyme (or inhibitor) and amine, may besimultaneously tested in a single step. That is, a user may use the kitto test a single sample for an enzyme (or inhibitor) and/or amine.

Referring to FIG. 1, for instance, one embodiment of an assay device 20that may be used in the present invention will now be described in moredetail. As shown, the assay device 20 contains a chromatographic medium23 optionally carried by a support 21. The chromatographic medium 23 maybe made from any of a variety of materials through which a fluid iscapable of passing, such as a fluidic channel, porous membrane, etc. Forexample, the chromatographic medium 23 may be a porous membrane formedfrom materials such as, but not limited to, natural, synthetic, ornaturally occurring materials that are synthetically modified, such aspolysaccharides (e.g., cellulose materials such as paper and cellulosederivatives, such as cellulose acetate and nitrocellulose); polyethersulfone; polyethylene; nylon; polyvinylidene fluoride (PVDF); polyester;polypropylene; silica; inorganic materials, such as deactivated alumina,diatomaceous earth, MgSO₄, or other inorganic finely divided materialuniformly dispersed in a porous polymer matrix, with polymers such asvinyl chloride, vinyl chloride-propylene copolymer, and vinylchloride-vinyl acetate copolymer; cloth, both naturally occurring (e.g.,cotton) and synthetic (e.g., nylon or rayon); porous gels, such assilica gel, agarose, dextran, and gelatin; polymeric films, such aspolyacrylamide; and so forth. In one particular embodiment, thechromatographic medium is formed from nitrocellulose and/or polyethersulfone materials. It should be understood that the term“nitrocellulose” refers to nitric acid esters of cellulose, which may benitrocellulose alone, or a mixed ester of nitric acid and other acids,such as aliphatic carboxylic acids having from 1 to 7 carbon atoms.

The support 21 may be formed from any material able to carry thechromatographic medium 23. Although not required, the support 21 may betransparent so that light readily passes therethrough. In addition, itis also generally desired that the support 21 is liquid-impermeable sothat fluid flowing through the medium does not leak through the support21. Examples of suitable materials for the support include, but are notlimited to, glass; polymeric materials, such as polystyrene,polypropylene, polyester (e.g., Mylar® film), polybutadiene,polyvinylchloride, polyamide, polycarbonate, epoxides, methacrylates,and polymelamine; and so forth. As is well known the art, thechromatographic medium 23 may be cast onto the support 21, wherein theresulting laminate may be die-cut to the desired size and shape.Alternatively, the chromatographic medium 23 may simply be laminated tothe support 21 with, for example, an adhesive. In some embodiments, anitrocellulose or nylon porous membrane is adhered to a Mylar® film. Anadhesive is used to bind the porous membrane to the Mylar® film, such asa pressure-sensitive adhesive. Laminate structures of this type arebelieved to be commercially available from Millipore Corp. of Bedford,Mass. Still other examples of suitable laminate structures are describedin U.S. Pat. No. 5,075,077 to Durley, III, et al., which is incorporatedherein in its entirety by reference thereto for all purposes.

The assay device 20 may also contain an absorbent material 28. Theabsorbent material 28 generally receives fluid that has migrated throughthe entire chromatographic medium 23. As is well known in the art, theabsorbent material 28 may assist in promoting capillary action and fluidflow through the medium 23. The assay device 20 may also include asample pad 22 or other material that is in fluid communication with thechromatographic medium 23. Some suitable materials that may be used toform the sample pad 22 include, but are not limited to, nitrocellulose,cellulose, porous polyethylene pads, and glass fiber filter paper. Ifdesired, the sample pad 22 may contain one or more assay pretreatmentreagents, either diffusively or non-diffusively attached thereto.

Generally speaking, the manner in which the assay device 20 functionsmay depend on the type of separation species selected for the reactivecomplexes. In this regard, various techniques for using the assay device20 in embodiments in which the separation species is a specific bindingmember will now be described in more detail. For example, as statedabove, the reactive complexes are generally allowed to incubate with thetest sample for a certain period of time. This incubation process may beconducted before applying the test sample to the chromatographic medium23, or it may be incorporated as part of the assaying procedure (i.e.,incubation occurs after the test sample is applied, such as within anincubation well). For instance, the incubation mixture may be directlyapplied to a portion of the chromatographic medium 23 through which itmay then travel in the direction illustrated by arrow “L” in FIG. 1.Alternatively, the mixture may first be applied to the sample pad 22.

Regardless, the chromatographic medium 23 defines a first enzymedetection zone 31 within which the separation species (e.g., specificbinding member) may be captured and detected. For example, in oneembodiment, a first receptive material is immobilized within the firstenzyme detection zone 31 that serves as a stationary binding site forreleased specific binding members, specific binding members present onunreacted complexes, or specific binding members present on partiallycleaved complexes. For example, in some embodiments, the first receptivematerial may be a biological receptive material. Such biologicalreceptive materials are well known in the art and may include, but arenot limited to, antibodies, antigens, haptens, biotin, avidin,streptavidin, neutravidin, captavidin, protein A, protein G,carbohydrates, lectins, nucleotide sequences, peptide sequences,effector and receptor molecules, hormone and hormone binding protein,enzyme cofactors and enzymes, enzyme inhibitors and enzymes, andderivatives thereof. As the enzyme concentration begins to increase inthe test sample, more reporters are released that have little or nospecific binding affinity for the receptive material at the first enzymedetection zone 31. The reduced quantity of reporters at the first enzymedetection zone 31 thus results in a decrease in signal intensity. Fromthis decrease in signal intensity, the presence or concentration of theenzyme may be readily determined. For example, in one embodiment, theamount of enzyme is inversely proportional to the signal intensity atthe first enzyme detection zone 31, I₁. If desired, the signal intensityI₁ may be plotted versus the enzyme concentration for a range of knownenzyme concentrations to generate an intensity curve. To determine thequantity of enzyme in an unknown test sample, the signal intensity maythen be converted to enzyme concentration according to the intensitycurve.

The first enzyme detection zone 31 may generally provide any number ofdistinct detection regions so that a user may better determine theconcentration of an enzyme within a test sample. Each region may containthe same or different receptive materials. For example, the enzymedetection zone 31 may include two or more distinct detection regions(e.g., lines, dots, etc.). The use of two or more distinct detectionregions may provide certain benefits, such as facilitatingsemi-quantitation and/or inhibiting potential false positives due tooverrunning of the reactive complexes or other materials. The detectionregions may be disposed in the form of lines in a directionsubstantially perpendicular to the flow of the test sample through thechromatographic medium 23. Likewise, in some embodiments, the detectionregions may be disposed in the form of lines in a directionsubstantially parallel to the flow of the test sample through the medium23. It should be understood that one or more distinct regions of thefirst enzyme detection zone 31 may exhibit the above-describedrelationship between signal intensity and enzyme concentration; however,each distinct region need not exhibit such a relationship. For example,in some embodiments, only one of multiple distinct regions may exhibit asignal intensity that is inversely proportional to the concentration ofthe enzyme. The signal intensity of other distinct regions, such asthose used to reduce false positives, may otherwise remain constant, orexhibit an increase and/or decrease in signal intensity. So long as atleast one distinct region of the enzyme detection zone 31 satisfies theinverse relationship, the signal intensity exhibited by the first enzymedetection zone 31 is considered inversely proportional to the enzymeconcentration.

The level of detection sensitivity for the enzyme of interest may beselectively controlled depending on the desired application. Oneparticular technique for controlling the detection sensitivity involvesmanipulating the quantity of the first receptive material used in thefirst enzyme detection zone 31. For instance, when assaying samplessuspected of containing large concentrations of an enzyme, the quantityof the first receptive material may be equal to or greater than theminimum required to capture the total quantity of reactive complexesutilized. Thus, if no enzyme were present in the test sample, all of thereactive complexes would become immobilized at the enzyme detection zone31 and all of the reporters would be present within the first enzymedetection zone 31. The minimum quantity required to capture all of thereactive complexes may be determined experimentally, and generallydepends upon the amount of the reactive complexes used and the bindingaffinity between the first receptive material and the specific bindingmember.

In applications where enhanced detection sensitivity is desired (e.g.,low suspected enzyme concentrations or short incubation times), thequantity of the first receptive material may be less than the minimumrequired to capture the total quantity of reactive complexes utilized.The use of such a limited quantity of the first receptive material mayprovide a variety of benefits, including decreasing the likelihood thatany partially cleaved complexes are captured at the first enzymedetection zone 31, which would otherwise result in a measured enzymeconcentration that is slightly lower than the actual concentration. Thatis, as the concentration of the enzyme increases, more specific bindingmembers are released from the reactive complexes. Due to their smallermolecular size, these released specific binding members generally reachthe first enzyme detection zone 31 faster than the partially cleavedcomplexes and unreacted complexes, and thus have a higher probability ofoccupying the available binding sites. Further, the partially cleavedcomplexes also generally contain a lesser quantity of specific bindingmembers than those that are completely unreacted. This reduction in thequantity of specific binding members statistically decreases the chancethat the partially cleaved complexes will bind to the first enzymedetection zone 31.

As discussed above, the inverse relationship between enzymeconcentration and signal intensity may be correlated to the actualenzyme concentration in the test sample. However, because it is notalways desirable to use an assay format that correlates an “increase” inenzyme concentration to a “decrease” in signal intensity (e.g. consumerapplications), the present invention also provides for embodiments inwhich an “increase” in enzyme concentration is directly correlated to an“increase” in signal intensity. In such cases, additional enzymedetection zones may be utilized. For example, referring again to FIG. 1,the chromatographic medium 23 may also define a second enzyme detectionzone 35 positioned downstream from the first enzyme detection zone 31.The second enzyme detection zone 35 may provide one or more distinctregions (e.g., line, dot, etc.), and may be positioned at anyorientation relative to the flow of the test sample.

Within the second enzyme detection zone 35, any released reporters,partially cleaved complexes, or unreacted complexes that do not bind tothe first receptive material at the enzyme detection zone 31 may becaptured and detected. The manner in which the released reporters arecaptured may depend on the nature of the reporters utilized. In someembodiments, a second receptive material may be immobilized within thesecond enzyme detection zone 35 for capturing reporters. For example,the second receptive material may be a biological receptive material,such as, but not limited to, antibodies, antigens, haptens, biotin,avidin, streptavidin, neutravidin, captavidin, protein A, protein G,carbohydrates, lectins, nucleotide sequences, peptide sequences,effector and receptor molecules, hormone and hormone binding protein,enzyme cofactors and enzymes, enzyme inhibitors and enzymes, andderivatives thereof. Because the second receptive material desirablybinds specifically to the reporters, it is normally different than thefirst receptive material.

For example, the released reporter may be conjugated with a specificbinding member selected to have an affinity for the second receptivematerial within the second enzyme detection zone 35. The specificbinding member may be conjugated to the reporter using any of a varietyof well-known techniques, such as through covalent bonding and/orphysical adsorption in a manner such as described above. In oneparticular embodiment, carboxylic groups of the reporter are activatedand reacted with amino groups of an antibody to form an amide bond. Inthis instance, the released reporter may become immobilized within thesecond enzyme detection zone 35 through specific binding between theantibody and a receptive material so that the signal generated by thedetectable substance may be detected. For example, the first receptivematerial may be a secondary antibody (such as an anti-biotin antibody,e.g., goat anti-Mouse IgG antibody), avidin (a highly cationic66,000-dalton glycoprotein), streptavidin (a nonglycosylated52,800-dalton protein), neutravid in (a deglysolated avid inderivative), or captavidin (a nitrated avidin derivative). In thisembodiment, the first receptive material may bind to a biotin (Mouse IgGantibody) specific binding member. The reporter may, for instance, be afluorescent-dyed particle conjugated with C-reactive protein, which maybind to a monoclonal antibody second receptive material (e.g., antiC-reactive protein (CRP) monoclonal antibody).

Of course, any other suitable technique for capturing and detection thereleased reporters may also be used. For example, in some embodiments,non-biological receptive materials may be immobilized within the secondenzyme detection zone 35 for capturing released reporters. Suchnon-biological receptive materials may be particularly useful incapturing, for example, released reporters that contain labeledparticles. For instance, in one embodiment, the receptive material is apolyelectrolyte. Polyelectrolytes may have a net positive or negativecharge, as well as a net charge that is generally neutral. Some suitableexamples of polyelectrolytes having a net positive charge include, butare not limited to, polylysine (commercially available fromSigma-Aldrich Chemical Co., Inc. of St. Louis, Mo.), polyethylenimine;epichlorohydrin-functionalized polyamines and/or polyamidoamines, suchas poly(dimethylamine-co-epichlorohydrin); polydiallyidimethyl-ammoniumchloride; cationic cellulose derivatives, such as cellulose copolymersor cellulose derivatives grafted with a quaternary ammoniumwater-soluble monomer; and so forth. In one particular embodiment,CelQuat® SC-230M or H-100 (available from National Starch & Chemical,Inc.), which are cellulosic derivatives containing a quaternary ammoniumwater-soluble monomer, may be utilized. Moreover, some suitable examplesof polyelectrolytes having a net negative charge include, but are notlimited to, polyacrylic acids, such as poly(ethylene-co-methacrylicacid, sodium salt), and so forth. It should also be understood thatother polyelectrolytes may also be utilized in the present invention,such as amphiphilic polyelectrolytes (i.e., having polar and non-polarportions). For instance, some examples of suitable amphiphilicpolyelectrolytes include, but are not limited to, poly(styryl-b-N-methyl2-vinyl pyridinium iodide) and poly(styryl-b-acrylic acid), both ofwhich are available from Polymer Source, Inc. of Dorval, Canada. Furtherexamples of polyelectrolytes are described in more detail in U.S. PatentApp. Publication No. 2003/0124739 to Song, et al., which is incorporatedherein in it entirety by reference thereto for all purposes.

Although any polyelectrolyte may generally be utilized, thepolyelectrolyte selected for a particular application may vary dependingon the nature of the released reporters. In particular, the distributedcharge of a polyelectrolyte allows it to bind to substances having anopposite charge. Thus, for example, polyelectrolytes having a netpositive charge are often better equipped to bind with releasedreporters (e.g., dyed particles) that are negatively charged, whilepolyelectrolytes that have a net negative charge are often betterequipped to bind to released reporters that are positively charged.Thus, in such instances, the ionic interaction between these moleculesallows the required binding to occur within the second enzyme detectionzone 35. Nevertheless, although ionic interaction is primarily utilizedto achieve the desired binding, it has also been discovered thatpolyelectrolytes may bind with reporters having a similar charge.

Besides using receptive materials, other capturing techniques may alsobe utilized. For example, in one embodiment, the reporter may contain amagnetic substance that is capable of being captured by a magneticdevice. In one embodiment, the magnetic device is positioned adjacent to(e.g., below) the second enzyme detection zone 35 defined by thechromatographic medium 23. In this manner, the magnetic device mayimmobilize the released reporters, as well as any partially cleaved orunreacted complexes, within the second enzyme detection zone 35. Anymagnetic device may be used in the present invention. A magnetic fieldgenerator, for instance, may be used to generate a magnetic field thatelicits a response from the magnetic substances. Suitable magnetic fieldgenerators include, but are not limited to, permanent magnets andelectromagnets. Some commercially available examples of suitablemagnetic separation devices include the Dynal MPC series of separatorsmanufactured by Dynal, Inc. of Lake Success, N.Y., which employ apermanent magnet located externally to a container holding a testmedium. Still other magnetic devices may be described in U.S. Pat. Nos.5,200,084 to Liberti, et al.; U.S. Pat. No. 5,647,994 to Tuunanen, etal.; U.S. Pat. No. 5,795,470 to Wang, et al.; and U.S. Pat. No.6,033,574 to Siddigi, which are incorporated herein in their entirety byreference thereto for all purposes.

When the reporters contain a substance that is directly detectable, anincrease in enzyme concentration results in an increase in the signalintensity at the second enzyme detection zone 35, I₂, due to thepresence of released reporters and/or partially cleaved complexes. Fromthis increase in signal intensity, the presence or concentration of theenzyme may be readily determined. For example, in one embodiment, theamount of enzyme is directly proportional to the signal intensity at thesecond enzyme detection zone 35, I₂. If desired, the signal intensity I₂may be plotted versus the enzyme concentration for a range of knownenzyme concentrations to generate an intensity curve. To determine thequantity of enzyme in an unknown test sample, the signal intensity maythen be converted to enzyme concentration according to the intensitycurve. It should be understood that, as discussed above with respect tothe first enzyme detection zone 31, so long as one distinct region ofthe second enzyme detection zone 35 satisfies the direct relationship,the signal intensity exhibited by the second enzyme detection zone 35 isconsidered directly proportional to the enzyme concentration.

Also, an inverse relationship may exist between the signal intensity atthe first enzyme detection zone 31 (I₁) and the second enzyme detectionzone 35 (I₂). For example, because a predetermined amount of reportersare present, the amount captured at the second enzyme detection zone 35is inversely proportional to the amount captured at the first enzymedetection zone 31. As a result of this inverse relationship, theconcentration of the enzyme may, in some cases, be more effectivelymeasured over an extended range by comparing the signal intensity atboth detection zones. For example, in one embodiment, the amount ofenzyme is directly proportional to the ratio of the signal intensity“I₂” to the signal intensity “I₁.” Based upon the range in which thisratio falls, the general concentration range for the enzyme may bedetermined. If desired, the ratio of I₂ to I₁ may be plotted versusenzyme concentration for a range of known enzyme concentrations togenerate an intensity curve. To determine the quantity of enzyme in anunknown test sample, the signal intensity ratio may then be converted toenzyme concentration according to the intensity curve. It should benoted that alternative mathematical relationships between I₁ and I₂ maybe plotted versus the enzyme concentration to generate the intensitycurve. For example, in one embodiment, the value of I₂/(I₂+I₁) may beplotted versus enzyme concentration to generate the intensity curve.

As stated above, certain embodiments of the present invention mayutilize a reporter that is not directly detectable. Thus, when released,it is generally desired that the reporter interact in some manner with adetectable substance for subsequent detection. For example, probescapable of generating a detectable signal may be employed that areconfigured to bind to the released reporters. For example, probes maycontain particles labeled or otherwise applied with the detectablesubstance. In some instances, it is desired to modify the probes in somemanner. For example, the probes may be modified with a specific bindingmember to form conjugated probes that have specific affinity for thereleased reporters. The specific binding members may generally beconjugated to the probes using any of a variety of well-knowntechniques, such as through covalent bonding and/or physical adsorptionin a manner such as described above. In one particular embodiment,carboxylic groups on the probe surface are activated and reacted withamino groups of the specific binding member to form an amide bond. Whenutilized, it is generally desired that the specific binding pair usedfor the probes and reporter is different than the specific binding pairused for the first receptive material and the other specific bindingmember joined to the substrate. This helps to ensure that the probes andreporters do not substantially interfere with the binding mechanismdescribed above.

The probes may be contacted with the released reporters at any stage ofthe enzyme detection process. For example, in some embodiments, theprobes may be applied to the assay device 20 at a location upstream fromthe region in which detection is desired. For example, in oneembodiment, the probes may be applied to a conjugated pad (not shown)that is located upstream from the enzyme detection zones 31 and 35, butdownstream from the sample pad 22.

In this embodiment, a variety of assay formats may be used to detect thereleased reporters. In one embodiment, for example, a “sandwich” assayformat is utilized in which the released reporter is selected to have anaffinity for the specific binding member of the conjugated probe. Thereleased reporter, such as antibodies, antigens, etc., typically has twoor more binding sites (e.g., epitopes). One of these binding sitesbecomes occupied by the specific binding member of the conjugated probe.However, the free binding site of the released reporter may subsequentlybind to a receptive material immobilized within the second enzymedetection zone 35 to form a new ternary sandwich complex. Alternatively,the released reporter may be detected using direct or indirect“competitive” assay formats. In such instances, the specific bindingmember of the conjugated probe may be the same as or an analog of thereleased reporter. Thus, upon reaching the second enzyme detection zone35, the conjugated detection probes and the released reporters competefor available binding sites of the immobilized receptive material. Ofcourse, any other assay format is also suitable for use in the presentinvention.

For the embodiments described above in which the reporters areindirectly detectable, an increase in enzyme concentration within thetest sample results in the release in a greater number of reporters.Thus, if a sandwich assay format is used, more released reporters bindto the conjugated probes so that the amount of enzyme is directlyproportional to the signal intensity at the second enzyme detection zone35. On the other hand, if a competitive assay format is used, the amountof enzyme is inversely proportional to the signal intensity at thesecond enzyme detection zone 35. In any event, the signal intensity maybe plotted versus the enzyme concentration for a range of known enzymeconcentrations to generate an intensity curve. To determine the quantityof enzyme in an unknown test sample, the signal intensity may then beconverted to enzyme concentration according to the intensity curve.

Besides techniques in which the separation species is chemicallyseparated from the reporter, other separation techniques are alsocontemplated in the present invention. For example, in one embodiment ofthe present invention, the separation species contains a magneticsubstance, such as described above. Thus, during and/or afterincubation, any released magnetic substances, partially cleaved reactivecomplexes, and unreacted complexes may be removed from the incubationmixture using a magnetic field. Once the magnetic substances (includingpartially cleaved and unreacted complexes) are removed, the remainingportion of the incubation mixture (e.g., the supernatant) may be testedfor the presence of an enzyme. Generally speaking, as enzymeconcentration begins to increase in the test sample, more reportersbecome released that are free from a magnetic substance and are thus notremoved during magnetic separation. Consequently, enzyme concentrationcorrelates to the quantity of the released reporters present in thesupernatant portion of the incubation mixture. If the reporter iscapable of directly generating a detection signal (e.g., luminescentcompounds, colored dyes, etc.), the presence or intensity of thedetection signal may simply be determined qualitatively, quantitatively,or semi-quantitatively. For example, in one embodiment, the amount ofenzyme is directly proportional to the signal intensity of the releasedreporters in the supernatant.

Typically, it is desired to assay the released reporters to determinethe presence or intensity of a detection signal. This may beparticularly useful in situations where the reporter is only indirectlydetectable (e.g., a specific binding member). Alternatively, even if thereporter is directly detectable, a subsequent assay may still enhancethe sensitivity and/or accuracy of detection. Any well-known assayingtechnique may be utilized to detect the reporters, including any of thetechniques described above and/or shown in FIG. 1. For example, thefirst enzyme detection zone 31 of FIG. 1 may be capable of capturing thereleased reporters. Specifically, the first enzyme detection zone 31 maycontain a receptive material having affinity for the reporter itself. Inanother embodiment, the first enzyme detection zone 31 may contain areceptive material that is capable of binding to binary complexes formedbetween the reporter and conjugated probes (“sandwich” assay format). Inboth cases, the amount of enzyme in the test sample is directlyproportional to the signal intensity at the first enzyme detection zone31.

Likewise, in still another embodiment, the first enzyme detection zone31 may contain a receptive material that has a specific binding affinityfor the reporter and conjugated probes, such that the reporter andconjugated probes compete for available binding sites (“directcompetitive” assay format). Alternatively, the first enzyme detectionzone 31 may contain a receptive material that has a specific bindingaffinity only for conjugated probes, and the reporter and receptivematerial compete for the binding sites of the conjugated probes(“indirect competitive” assay format). In both cases, the amount ofenzyme in the test sample is inversely proportional to the signalintensity at the first enzyme detection zone 31. If desired, asdiscussed above, a second enzyme detection zone 35 may also be utilized.For example, in embodiments in which magnetic separation is employed,the second enzyme detection zone 35 may be capable of capturing anyconjugated probes that do not bind to the first enzyme detection zone31. Thus, in such embodiments, the amount of enzyme in the test sampleis directly proportional to the signal intensity at the second enzymedetection zone 35.

In the embodiments described above, magnetic separation of the releasedmagnetic substance, partially cleaved reactive complexes, and unreactedcomplexes, may occur prior to assaying the released reporters. In someembodiments, however, the magnetic separation step may be incorporatedas part of the assaying procedure. For instance, referring again to FIG.1, a magnetic device (not shown) may be positioned adjacent to themedium 23 at a location at or near (e.g., downstream) the point ofapplication, e.g., the sample pad 22. Thus, when the incubation mixtureflows through the medium 23, any magnetic substances (released magneticsubstances, partially cleaved reactive complexes, and/or unreactedcomplexes) become immobilized within a separation zone. The magneticdevice may also be positioned upstream from a point in which conjugatedprobes are optionally contacted with the released reporters (e.g., aconjugate pad). The reporters, having been separated from the magneticsubstances, may then be assayed using detection zones 31 and/or 35 asdescribed above.

The aforementioned detection techniques are described specifically inthe context of enzymes. However, as stated, the present invention isequally suitable for detecting the presence or quantity of an enzymeinhibitor within a test sample. To detect the presence of an enzymeinhibitor within a test sample, a predetermined quantity of acorresponding enzyme may be mixed with the test sample and allowed toincubate. In the presence of a certain amount of an enzyme inhibitor,the enzyme-catalyzed reaction does not proceed at a detectable rate.Thus, the relationship between enzyme inhibitor concentration and signalintensity will be opposite to the relationship between enzymeconcentration and signal intensity. As an illustration, anenzyme-catalyzed reaction will not occur in the presence of a certainamount of inhibitor. Thus, in one embodiment, all of the reactivecomplexes will be captured at the enzyme detection zone 31, whichgenerates its maximum signal intensity. On the other hand, as the amountof enzyme inhibitor is reduced, the enzyme causes the reporters torelease from the reactive complexes as described above. The signalintensity generated at the first enzyme detection zone 31 thus begins todecrease due to a corresponding decrease in the presence of releasedreporters. Likewise, the signal intensity generated at the second enzymedetection zone 35 may, in some embodiments, begin to increase due to acorresponding increase in the presence of released reporters.Accordingly, in this particular embodiment, the amount of enzymeinhibitor within the test sample is inversely proportional to the signalintensity at the first enzyme detection zone 31 and directlyproportional to the signal intensity at the second enzyme detection zone35.

II. Amine Detection

Amine detection may be accomplished in a variety of different ways. Inone embodiment, for example, amine detection is accomplished in thepresent invention using a “chemichromic dye”, i.e., a dye that exhibitsa detectable color change upon chemical reaction with one or morefunctional groups. Without intending to be limited by theory, it isbelieved that the addition of an amino functional group (NH₂) to thechemichromic dye molecule induces either a shift of the absorptionmaxima towards the red end of the spectrum (“bathochromic shift”) ortowards the blue end of the spectrum (“hypsochromic shift”). The type ofabsorption shift depends on the nature of the dye molecule and onwhether the amino group functions as an electron acceptor (oxidizingagent), in which a hypsochromic shift results, or whether the aminogroup functions as an electron donor (reducing agent), in which abathochromic shift results. Regardless, the absorption shift provides acolor difference that is detectable, either visually or throughinstrumentation, to indicate the presence of amines in the test sample.For example, prior to contact with an infected test sample, thechemichromic dye may be colorless or it may possess a certain color.However, after contacting the test sample and reacting with aminespresent therein, the dye exhibits a change in color that is differentthan its initial color. That is, the dye may change from a first colorto a second color, from no color to a color, or from a color to nocolor.

Generally speaking, any chemichromic dye capable of exhibiting adetectable change in color upon reaction with an amine may be utilizedin the present invention. Such dyes are generally well known to thoseskill in the art, and may be described, for instance, in U.S. Pat. Nos.4,477,635 to Mitra; U.S. Pat. No. 5,837,429 to Nohr, et al.; U.S. Pat.No. 6,174,646 to Hirai, et al., which are incorporated herein in theirentirety by reference thereto for all purposes. For example, one classof chemichromic dyes that is particularly useful in the presentinvention is arylmethane dyes, such as diarylmethanes, triarylmethanes,and so forth.

Triarylmethane dyes, for example, may have the following generalstructure:

wherein R, R′, and R″ are independently selected from substituted andunsubstituted aryl groups, such as phenyl, naphthyl, anthracenyl, etc.The aryl groups may, for example, be substituted with functional groups,such as amino, hydroxyl, carbonyl, carboxyl, sulfonic, alkyl, and/orother known functional groups. When contacted with the dye, the aminogroup of the amine (e.g., ammonia, diamines, and/or tertiary amines)reacts with the central carbon atom of the dye. The addition of theamino group causes the dye to undergo a change in color. An example ofthe resulting structure is set forth below:

One particular example of a suitable triarylmethane dye is pararosanilin(also known as “basic fuchsin” or “magenta 0”) and analogs thereof, suchas rosanilin (“magenta I”), magenta II, new fuchsin (“magenta III”),methyl violet 2B, methyl violet 6B, methyl violet 10B (“crystalviolet”), methyl green, ethyl green, acid fuchsin, and so forth.Pararosanilin shifts from a red color to colorless (i.e., white) uponreaction with an amine. Pararosanilin contains three phenylamine groups(i.e., amino-substituted aryl groups). Specifically, the structure ofthe structure of pararosanilin is set forth below:

In some cases, triarylmethane dyes may be formed by converting a leucobase to a colorless carbinol and then treating the carbinol with an acidto oxidize the carbinol and form the dye. Thus, for example,pararosanilin may be derived by reacting the carbinol form ofpararosanilin (“pararosaniline base”) with an acid, such as, but notlimited to, sulfonic acids, phosphoric acids, hydrochloric acid, and soforth. The carbinol form of pararosanilin is set forth below.

Another example of a suitable triarylmethane dye isalpha-naphtholbenzein and analogs thereof. Alpha-naphtholbenzein turnsfrom an orange/red color to a gray/black color upon reaction with anamine. Alpha-naphtholbenzein contains a hydroxyl-substituted naphthylgroup, a carbonyl-substituted naphthyl group, and a phenyl group.Specifically, the structure of alpha-naphtholbenzein is set forth below:

Still another example of a suitable triarylmethane dye is naphthocromegreen and analogs thereof. Naphthocrome green turns from a pale yellowcolor to a blue/green color upon reaction with an amine. Similar toalpha-naphtholbenzein, naphthocrome green contains ahydroxyl-substituted naphthyl group, a carbonyl-substituted naphthylgroup, and a phenyl group. However, each naphthyl group is alsosubstituted with a sodium carboxyl. Specifically, the structure ofnaphthocrome green is set forth below:

As indicated above, diarylmethanes may also be used in the presentinvention. One example of such a diarylmethane is 4,4′-bis(dimethylamino) benzhydrol (also known as “Michler's hydrol”), which hasthe following structure:

Still other examples include analogs of Michler's hydrol, such asMichler's hydrol leucobenzotriazole, Michler's hydrol leucomorpholine,Michier's hydrol leucobenzenesulfonamide, and so forth, as well as otherdiarylmethanes, such as malachite green leuco, malachite green carbinol,sodium 2,6-dichloroindopheno-late, rhodamine lactam, crystal violetlactone, and crystal violet leuco.

Generally speaking, any of a variety of devices may be utilized thatemploy that amine detection techniques of the present invention. Forexample, in one embodiment, an assay device may be utilized thatincorporates an amine detection zone. Referring again to FIG. 1, forinstance, the chromatographic medium 23 may define an amine detectionzone 61 within which is contained a chemichromic dye. The aminedetection zone 61 may generally be located downstream or upstream fromthe first enzyme detection zone 31 and/or the second enzyme detectionzone 35. In the illustrated embodiment, for example, the amine detectionzone 61 is located downstream from both the first enzyme detection zone31 and the second enzyme detection zone 33. Although not required, thisparticular configuration may help reduce the likelihood that any enzymeor enzyme inhibitors within the test sample inadvertently react with thechemichromic dye.

The dye may be applied to the chromatographic medium 23 in a variety ofdifferent ways. For example, the dye may be applied directly to themedium 23 or first formed into a solution prior to application. Varioussolvents may be utilized to form the solution, such as, but not limitedto, acetonitrile, dimethylsulfoxide (DMSO), ethyl alcohol,dimethylformamide (DMF), and other polar organic solvents. The amount ofthe dye in the solution may range from about 0.001 to about 1 milligramper milliliter of solvent, and in some embodiments, from about 0.01 toabout 0.1 milligrams per milliliter of solvent. The dye solution may becoated onto the chromatographic medium 23 using well-known techniquesand then dried. The dye concentration may be selectively controlled toprovide the desired level of detection sensitivity. Higherconcentrations may provide a higher level of detection sensitivity whenlow amine levels are suspected. For example, as is well known in theart, the amount of the dye present within the amine detection zone 61may be tailored to be equal to or in excess of the maximum amount ofsuspected amines within the test sample.

The chemichromic dye may be applied in a manner so that it does notsubstantially diffuse through the matrix of the chromatographic medium23. This enables a user to readily detect the change in color thatoccurs upon reaction of the dye with an amine. For instance, thechemichromic dye may form an ionic and/or covalent bond with functionalgroups present on the surface of the chromatographic medium 23 so thatit remains immobilized thereon. For example, in one embodiment, apositively-charged chemichromic dye may form an ionic bond withnegatively-charged carboxyl groups present on the surface of some porousmembranes (e.g., nitrocellulose). In other embodiments, the use ofparticles may facilitate the immobilization of the chemichromic dye atthe amine detection zone 61. Namely, the dye may be coated ontoparticles, such as described above, which are then immobilized on thechromatographic medium 23. In this manner, the dye is able to readilycontact a test sample flowing through the medium 23.

Although non-diffusive immobilizing techniques may be desired in somecases, it should also be understood that any other technique forapplying the chemichromic dye to the chromatographic medium 23 may beused in the present invention. In fact, the aforementioned methods areonly intended to be exemplary of the techniques that may be used in thepresent invention. For example, in some embodiments, certain componentsmay be added to a chemichromic dye solution that substantially inhibitthe diffusion of the dye into the matrix of the chromatographic medium23. In other cases, immobilization may not be required, and the dye mayinstead diffuse into the matrix of the chromatographic medium 23 forreaction with the test sample.

The amine detection zone 61 may generally provide any number of distinctdetection regions so that a user may better determine the concentrationof an amine within the test sample. Each region may contain thechemichromic dye, or may contain different dyes for reacting withdifferent types of amines. For example, the amine detection zone 61 mayinclude two or more distinct detection regions (e.g., lines, dots,etc.). The detection regions may be disposed in the form of lines in adirection substantially perpendicular to the flow of the test samplethrough the chromatographic medium 23. Likewise, in some embodiments,the detection regions may be disposed in the form of lines in adirection substantially parallel to the flow of the test sample throughthe assay device.

III. Detection Techniques

After the assaying procedure, the first enzyme detection zone 31, thesecond enzyme detection zone 35, and/or the amine detection zone 61 maybe analyzed, either qualitatively (e.g., visual observation), orsemi-quantitatively or quantitatively (e.g., using instrumentation), forthe presence or intensity of a respective signal. Depending on thenature of the reporters and the chemichromic dye utilized, the same ordifferent detection techniques may be employed for the amine detectionzone 61 as the enzyme detection zones 31 and/or 35. For example,reflectance techniques may be utilized for the amine detection zone 61and enzyme detection zones 31 and/or 35 to quantitatively orsemi-quantitatively determine color or color intensity. In oneembodiment, color intensity of dyed reporters and a chemichromic dye maybe measured as a function of absorbance, with an increased absorbancegenerally representing an increased amine concentration. For example,absorbance readings may be measured at a wavelength of 650 nanometersusing a microplate reader from Dynex Technologies of Chantilly, Va.(Model # MRX). In still another embodiment, color intensity may bemeasured using a conventional test known as “CIELAB”, which is discussedin Pocket Guide to Digital Pr inting by F. Cost, Delmar Publishers,Albany, N.Y. ISBN 0-8273-7592-1 at pages 144 and 145. This methoddefines three variables, L*, a*, and b*, which correspond to threecharacteristics of a perceived color based on the opponent theory ofcolor perception. The three variables have the following meaning:

-   -   L*=Lightness, ranging from 0 to 100, where 0=dark and 100=light;    -   a*=Red/green axis, ranging approximately from −100 to 100;        positive values are reddish and negative values are greenish;        and    -   b*=Yellow/blue axis, ranging approximately from −100 to 100;        positive values are yellowish and negative values are bluish.

Because CIELAB color space is somewhat uniform, a single number may becalculated that represents the difference between two colors asperceived by a human. This difference is termed ΔE and calculated bytaking the square root of the sum of the squares of the threedifferences (ΔL*, Δa*, and Δb*) between the two colors. In CIELAB colorspace, each ΔE unit is approximately equal to a “just noticeable”difference between two colors. CIELAB is therefore a good measure for anobjective device-independent color specification system that may be usedas a reference color space for the purpose of color management andexpression of changes in color. Using this test, color intensities (L*,a*, and b*) may thus be measured using, for instance, a handheldspectrophotometer from Minolta Co. Ltd. of Osaka, Japan (Model #CM2600d). This instrument utilizes the D/8 geometry conforming to CIENo. 15, ISO 7724/1, ASTME1164 and JIS Z8722-1982 (diffusedillumination/8-degree viewing system. The D65 light reflected by thespecimen surface at an angle of 8 degrees to the normal of the surfaceis received by the specimen-measuring optical system. Still othersuitable devices for measuring the intensity of a visual color may alsobe used in the present invention. For example, a suitable reflectancespectrophotometer or reader that may be used in the present invention isdescribed in U.S. Patent App. Pub. No. 2003/0119202 to Kaylor, et al.,which is incorporated herein in its entirety by reference thereto forall purposes.

In some cases, the detection techniques used for the amine detectionzone 61 may be different than those used for the enzyme detection zones31 and 35. For example, in one embodiment, the signal intensity of theenzyme detection zones 31 and 35 may be measured using fluorescencedetection techniques. Fluorescence detection generally utilizeswavelength filtering to isolate the emission photons from the excitationphotons, and a detector that registers emission photons and produces arecordable output, usually as an electrical signal or a photographicimage. One suitable fluorescence detector for use with the presentinvention is a FluoroLog III Spectrofluorometer, which is sold by SPEXIndustries, Inc. of Edison, N.J. Another example of a suitablefluorescence detector is described in U.S. Patent ApplicationPublication No. 2004/0043502 to Song, et al., which is incorporatedherein in its entirety by reference thereto for all purposes. Stillother known detection techniques may also be utilized in the presentinvention for the first enzyme detection zone 31, the second enzymedetection zone 35, and/or the amine detection zone 61. For example,other suitable optical detection techniques may include, but not limitedto, phosphorescence, diffraction, transmittance, etc. An optical readermay be capable of emitting light and also registering a detection signal(e.g., transmitted or reflected light, emitted fluorescence orphosphorescence, etc.).

Regardless of the manner in which the detection zones are analyzed, theresult may be compared with a predetermined detection curve in which thesignal (e.g., color or color intensity) of the first enzyme detectionzone 31, the second enzyme detection zone 35, and/or the amine detectionzone 61, is plotted versus various known concentrations of an enzyme,enzyme inhibitor, or amines. In this manner, the signal intensity may bemeasured and readily correlated to a certain concentration for providingquantitative or semi-quantitative results to a user.

Referring to FIG. 3, one particular embodiment of a method for detectingthe presence of a protease and amine will now be described in moredetail. Initially, a test sample containing a protease P and amine A ismixed with reactive complexes 41 that each include a dyed particle 43and specific binding member 45 (e.g., biotinylated substance) joined toa substrate 47 (e.g., protein or glycoprotein). The complexes 41 areallowed to incubate for a sufficient period of time to form anincubation mixture (designated numeral 65 in FIG. 3) that includesreleased dyed particles 43 and specific binding members 45, as well asan unreacted complex 41, partially cleaved complex 49, protease P, amineA, and any product (not shown) generated by the enzyme-catalyzedreaction. In some cases, the amine A may slightly inhibit theenzyme-catalyzed reaction; however, it is not believed that any suchinhibition will have a significant affect on the accuracy of the assay.

The incubation mixture 65 is applied to the sample pad 22, as indicatedby the illustrated directional arrow, and then travels to the firstenzyme detection zone 31. Due to their smaller size, the releasedspecific binding members 45 flow faster and have a higher probability ofbeing captured by a first receptive material 90 within the first enzymedetection zone 31. The available binding sites in the first enzymedetection zone 31 may also be occupied by some of the unreactedcomplexes 41 and partially cleaved complexes 49. However, any unreactedcomplexes 41 and partially cleaved complexes 49 not captured by thefirst enzyme detection zone 31 travel to the second enzyme detectionzone 35 and bind to a receptive material (not shown) contained therein.Because the released dyed particles 43 do not have an affinity for thefirst receptive material 90, they also travel to the second enzymedetection zone 35 and bind to a receptive material (not shown) containedtherein. In addition, because the amines A have no specific affinity forthe receptive materials within the first and second enzyme detectionzones 31 and 35, they travel to the amine detection zone 61 where theyreact with the chemichromic dye (not shown). After the reaction, theamine detection zone 61 changes color in a manner that is detectableeither visually or with instrumentation.

The present invention provides a relatively simple, compact andcost-efficient device for accurately detecting enzymes (or enzymeinhibitors) and/or amines within a test sample (e.g., vaginal fluid). Inthis manner, for example, vaginal fluid may be tested in a single stepfor the presence of amines and also for the presence of other diseasesor disorders. The test result may be visible so that it is readilyobserved by the person performing the test in a prompt manner and undertest conditions conducive to highly reliable and consistent testresults. The device may then be discarded as a unit when the test isconcluded. Such a single step detection technique has a variety ofbenefits. For example, as discussed above, the detection of amineswithin a vaginal fluid test sample may indicate the presence of certaintypes of vaginal infection (e.g., bacterial vaginosis or trichomonasvaginitis), while the detection of an enzyme or enzyme inhibitor withinthe test sample may indicate the presence of other types of vaginalinfection (e.g., candidal vaginitis).

Besides diagnosing one or more types of infection in vaginal fluid, themethod and diagnostic kit of the present invention may be used in anyother application in which the detection of an enzyme, enzyme inhibitor,and/or amine may be desired. For example, many people (e.g., diabetics,burn victims, those suffering from suppressed immune systems, etc.) whohave difficulty in healing and require extended periods for proper andcomplete wound healing are susceptible to infection. Bacteria and moldmay also cause infection in hosts other than the human body, such asfood. In many cases, these infections result in the formation of odorousamines and diamines, which may be produced by the metabolic processes ofproteolytic bacteria together with short chain organic acids. Thus, aswith vaginal infections, the ability to detect amines in other typescontexts, such as in a wound exudate or food, may prove vastlybeneficial. Likewise, the mere presence of an enzyme may, in some cases,indicate the existence of tissue or organ damage. Abnormal enzymeconcentrations may also indicate other conditions, such as a bacterialor viral infection. For instance, the presence or concentration of anenzyme in a test sample may also serve as a diagnostic marker for sometypes of cancers and other conditions. As an example, prostate-specificantigen (PSA) is a well-known marker for prostate cancer. Other examplesof diagnostic markers include cathepsin B (cancer), cathepsin G(emphysema, rheumatoid arthritis, inflammation), plasminogen activator(thrombosis, chronic inflammation, cancer), and urokinase (cancer).

The present invention may be better understood with reference to thefollowing examples.

EXAMPLE 1

β-casein was initially conjugated to dyed particles. Specifically, 2milliliters of blue carboxylated particles (0.3-micrometer particlesize, Bangs Laboratories, Inc. of Fisher, Ind.) were washed once withphosphate-buffered saline (PBS from Polysciences, Inc. of Warrington,Pa.) and then suspended in 1 milliliter of PBS. 36 milligrams ofcarbodiimide (Polysciences, Inc.) in 1 milliliter of PBS was added andthe mixture was shaken for 30 minutes. The particles were washed twicewith a borate buffer (Polysciences, Inc.), and then suspended in 1milliliter of borate buffer. 1 milligram of β-casein (Sigma-AldrichChemical Co., Inc. of St. Louis, Mo.) was added and the mixture wasshaken overnight at room temperature. The particles were washed oncewith the borate buffer and then re-suspended in 500 microliters ofborate buffer. 1 milliliter of ethanolamine solution (0.1 molar,Polysciences Inc.) was added to the particles and shaken for 30 minutes.The particles were then washed four times with water and suspended in 2milliliters of borate buffer.

Upon formation, the conjugated particles (hereinafter “BP-casein”) werethen biotinylated. Specifically, 4 milligrams of the BP-casein particlesin 200 microliters of borate buffer were mixed with 1 milligram ofEZ-Link® Sulfo-NHS-LC-Biotin (Pierce Biotechnology, Inc. of Rockford,Ill.) in 200 microliters of borate buffer. The mixture was shakenovernight and then washed five times with water. The washed particleswere suspended in 1 milliliter of tris buffer (pH of 7.2, 20millimolar). The biotinylated conjugated particles are hereinafterreferred to as “BP-casein-B.”

EXAMPLE 2

The ability to form a membrane-based device for amine and/or enzymeassays was demonstrated. Initially, Millipore HF12002 porousnitrocellulose membranes were laminated onto corresponding supportingcards having a length of approximately 30 centimeters. Streptavidin (1.0milligram per milliliter, Sigma-Aldrich Chemical Co., Inc.) was stripedonto the membrane to form a first enzyme detection zone and Goldline™ (apolylysine solution obtained from British Biocell International) wasstriped onto the membrane (downstream from the first enzyme detectionzone) to form a second enzyme detection zone. Alpha-naphtholbenzein(ANB) (5 milligrams per milliliter, Sigma-Aldrich Chemical Co., Inc.)was also striped onto the membrane (downstream from the enzyme detectionzones) to form an amine detection zone. The membrane was dried for 1hour at 37° C. A cellulosic fiber wicking pad (Millipore Co.) wasattached to the end of the membrane closest to the amine detection zone.The assembled card was then cut into 4-millimeter wide devices. Theresulting devices were sealed in a bag for storage.

EXAMPLE 3

The ability to form a membrane-based device for amine and/or enzymeassays was demonstrated. Initially, Millipore HF12002 porousnitrocellulose membranes were laminated onto corresponding supportingcards having a length of approximately 30 centimeters. Anti-biotinantibody (2.0 milligrams per milliliter, Sigma-Aldrich Chemical Co.,Inc.) was striped onto the membrane to form a first enzyme detectionzone and Goldline™ (a polylysine solution obtained from British BiocellInternational) was striped onto the membrane (downstream from the firstenzyme detection zone) to form a second enzyme detection zone. ANB dye(Sigma-Aldrich Chemical Co., Inc.) was also striped onto the membrane(downstream from the enzyme detection zones) to form an amine detectionzone. The membrane was dried for 1 hour at 37° C. A cellulosic fiberwicking pad (Millipore Co.) was attached to the end of the membraneclosest to the ANB dye zone. The assembled card was then cut into4-millimeter wide devices. The resulting devices were sealed in a bagfor storage.

EXAMPLE 4

The ability to detect the presence of an enzyme and amine in accordancewith the present invention was demonstrated. Four samples were tested.Each sample contained 13 microliters of the “BP-casein-B” of Example 1(10 milligrams per milliliter); 100 microliters of Tween 20 (2%,Sigma-Aldrich Chemical Co., Inc.); and 300 microliters of tris buffer(pH of 7.4). Samples 1-4 contained cadaverine in an amount of 0.2, 0.0,0.2, and 0.2 milligrams, respectively. Samples 1-4 also contained anactive protease from Bacillus polymyxa (20 milligrams/milliliter), whichis a metalloenzyme available from Sigma-Aldrich Chemical Co., Inc., inan amount of 0.5, 0.5, 0, and 0.05 micrograms, respectively. Sample 2(the amine control) also contained 10 additional microliters of trisbuffer (pH of 7.4), and Sample 3 (the enzyme control) contained 5additional microliters of tris buffer (pH of 7.4). The samples wereallowed to incubate for 10 minutes.

Each sample was then transferred to a well present on a microtiterplate. The assay device samples of Example 2 were then inserted intoeach respective well to initiate the test. After allowing the assay todevelop for 10 minutes, the color interisity of each detection zone wasobserved. The qualitative results are set forth below in Table 1.

TABLE 1 Qualitative Color Results for Detection Zones First EnzymeSecond Enzyme Amine Sample Detection Zone Detection Zone Detection Zone1 Weak Strong Gray 2 None Strong Yellow 3 Strong None Gray 4 StrongMedium Gray

As indicated, the signal intensity exhibited by the first enzymedetection zone decreased in the presence of the enzyme, while the signalintensity exhibited by the second detection zone increased in thepresence of the enzyme. The color of the amine detection zone changedfrom yellow to gray in the presence of the amine.

EXAMPLE 5

The ability to detect the presence of an enzyme and amine in accordancewith the present invention was demonstrated. Eight samples were tested.Each sample contained 25 microliters of the “BP-casein-B” of Example 1(10 milligrams per milliliter); 600 microliters of Tween 20 (2%,Sigma-Aldrich Chemical Co., Inc.); and 900 microliters of Hepes buffer(pH of 7.2). Samples 1-8 contained cadaverine in an amount of 0.000(amine control), 0.234, 0.468, 0.937, 1.875, 3.750, 7.500 and 15.000micrograms per milliliter, respectively. Samples 1-8 also contained anactive protease from Bacillus polymyxa, which is a metalloenzymeavailable from Sigma-Aldrich Chemical Co., Inc., in an amount of 0.0000(enzyme control), 0.0275, 0.0550, 0.1100, 0.2200, 1.1000, 2.2000 and11.0000 micrograms per milliliter, respectively. The samples wereallowed to incubate for 10 minutes.

Each sample was then transferred to a well present on a microtiterplate. The assay device samples of Example 3 were then inserted intoeach respective well to initiate the test. After allowing the assay todevelop for 10 minutes, the color intensity of each detection zone wasobserved. The qualitative results are set forth below in Table 2.

TABLE 2 Qualitative Color Results for Detection Zones First EnzymeSecond Enzyme Amine Sample Detection Zone Detection Zone Detection Zone1 Strong None Yellow 2 Strong None Yellow 3 Strong Weak Yellow/Gray 4Strong Medium Yellow/Gray 5 Medium Medium Yellow/gray 6 Medium StrongYellow/gray 7 Weak Strong Gray 8 None Strong Gray

As indicated, the signal intensity exhibited by the first enzymedetection zone decreased in the presence of the enzyme, while the signalintensity exhibited by the second enzyme detection zone increased in thepresence of the enzyme. The color of the amine detection zone changedfrom yellow to gray in the presence of the amine.

EXAMPLE 6

The ability to detect the presence of an enzyme and amine in accordancewith the present invention was demonstrated. Six samples were tested.Each sample contained 50 micrograms of the “BP-casein-B” of Example 1;Tween 20 (2%, Sigma-Aldrich Chemical Co., Inc.); and 40 microliters oftris buffer (pH of 7.4). Samples 1-6 contained cadaverine in an amountof 0.00 (amine control), 0.30, 0.60, 1.25, 2.50 and 10.0 milligrams permilliliter, respectively. Samples 1-6 also contained an active proteasefrom Bacillus polymyxa, which is a metalloenzyme available fromSigma-Aldrich Chemical Co., Inc., in an amount of 0, 1, 2, 4, 8 and 40nanograms per milliliter, respectively. The samples were allowed toincubate for 10 minutes.

Each sample was then transferred to a well present on a microtiterplate. The assay device samples of Example 2 were then inserted intoeach respective well to initiate the test. After allowing the assay todevelop for 10 minutes, the reflectance intensity of each detection zonewas measured using a reflectance reader. The quantitative results areset forth below in Table 3.

TABLE 3 Quantitative Color Intensity for Detection Zones ReflectanceReflectance Intensity (I₁) Intensity (I₂) Amine of First of SecondDetection Sample Detection Zone Detection Zone Zone 1 2.0490 0.02630.2687 2 1.4070 0.6988 0.2890 3 1.1980 1.0920 0.2918 4 1.1080 1.33200.3169 5 0.8213 1.2800 0.3696 6 0.4298 1.2140 0.7020

As indicated, the signal intensity exhibited by the first enzymedetection zone decreased in the presence of the enzyme, while the signalintensity exhibited by the second enzyme detection zone increased in thepresence of the enzyme. Also, the intensity of the amine detection zoneincreased in the presence of amine.

While the invention has been described in detail with respect to thespecific embodiments thereof, it will be appreciated that those skilledin the art, upon attaining an understanding of the foregoing, mayreadily conceive of alterations to, variations of, and equivalents tothese embodiments. Accordingly, the scope of the present inventionshould be assessed as that of the appended claims and any equivalentsthereto.

1. A diagnostic kit for detecting an amine or a hydrolytic enzyme withina test sample, the kit comprising: a plurality of reactive complexesthat each comprises a substrate joined to a reporter and a specificbinding member, said substrate being cleavable by a hydrolytic enzyme torelease said reporter; and a chromatographic medium for the receipt ofsaid reactive complexes, reporters, or both, said chromatographic mediumdefining a first enzyme detection zone within which is immobilized areceptive material that has an affinity for said specific binding memberfor generating an enzyme detection signal, wherein the presence orquantity of a hydrolytic enzyme is determinable from said enzymedetection signal, said chromatographic medium further defining an aminedetection zone positioned downstream from said first enzyme detectionzone, wherein a chemichromic dye is contained within said aminedetection zone, said chemichromic dye being capable of undergoing acolor change in the presence of an amine, wherein the presence orquantity of an amine is determinable from said color change.
 2. Adiagnostic test kit as defined in claim 1, wherein said reportercomprises a detectable substance that is capable of directly generatingsaid enzyme detection signal.
 3. A diagnostic test kit as defined inclaim 1, wherein the enzyme is a protease or peptidase.
 4. A diagnostictest kit as defined in claim 1, wherein said substrate is a protein,glycoprotein, peptide, nucleic acid, carbohydrate, lipid, ester, orderivative thereof.
 5. A diagnostic test kit as defined in claim 1,wherein said substrate is casein, albumin, hemoglobin, myoglobin,keratin, gelatin, insulin, proteoglycan, fibronectin, lamin in,collagen, elastin, or a derivative thereof.
 6. A diagnostic test kit asdefined in claim 1, wherein said chemichromic dye is an arylmethane. 7.A diagnostic test kit as defined in claim 1, wherein said chemichromicdye is a triarylmethane having the following general structure:

wherein R, R′, and R″ are independently selected from substituted andunsubstituted aryl groups.
 8. A diagnostic test kit as defined in claim1, wherein said chemichromic dye is a diarylmethane.
 9. A diagnostictest kit as defined in claim 1, wherein said chromatographic mediumcontains a porous membrane.
 10. A diagnostic test kit as defined inclaim 1, wherein said reporter comprises a specific binding member. 11.A diagnostic test kit as defined in claim 10, further comprising probesconjugated with a specific binding member, said probes comprising adetectable substance that is capable of directly generating said enzymedetection signal.
 12. A diagnostic test kit as defined in claim 1,wherein said specific binding member is immunoreactive.
 13. A diagnostictest kit as defined in claim 12, wherein said specific binding memberincludes an antibody, antigen, hapten, or a derivative thereof.
 14. Adiagnostic test kit as defined in claim 1, wherein said receptivematerial is immunoreactive.
 15. A diagnostic test kit as defined inclaim 14, wherein said receptive material includes an antibody, antigen,hapten, or a derivative thereof.
 16. A diagnostic test kit as defined inclaim 1, wherein said receptive material includes an anti-biotinantibody and said specific binding member includes biotin.
 17. Adiagnostic test kit as defined in claim 1, wherein said chromatographicmedium further comprises a second enzyme detection zone capable ofcapturing said reporter or complexes thereof to generate a second enzymedetection signal.
 18. A diagnostic test kit as defined in claim 17,wherein a second receptive material is immobilized within said seconddetection zone that is capable of binding to probes or complexes thereofto generate said second enzyme detection signal.